This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A key question in understanding ubiquitin (Ub) conjugation is how Ub is transferred between enzymes in E1-E2-E3 cascades. For E3s in the HECT (Homologous to E6AP C-Terminus) class, the ~40 kDa C-terminal HECT domain binds a reactive thioester-linked E2~Ub (here "~" refers to thioester or thioester-like covalent linkage). Then a transthiolation reaction ensues, whereby Ub is transferred from the E2 catalytic Cys to the HECT domain catalytic Cys. Thus, the Ub C-terminus and the active sites of the E2 and HECT domain must all be juxtaposed for E2-to-E3 Ub transfer. In humans, nearly 30 HECT E3s become charged by selective interactions with distinct E2s, and subsequently catalyze target ubiquitination. For example, the HECT E3 NEDD4L has been shown to bind and receive Ub from a subset of E2s including UbcH5B and Ube2E3. A well-recognized downstream function of NEDD4L is regulation of blood pressure through ubiquitination of the Epithelial Sodium Channel (ENaC). Despite important physiological roles of NEDD4L and other HECT E3s, their fundamental enzymatic mechanisms remain poorly understood. A particularly vexing question is how a HECT domain and a specific Ub-loaded E2 interact to promote Ub transfer. Prior studies showed that HECT domains have two structural "lobes" tethered by a flexible linker. The N-terminal "N-lobe" binds part of an E2 distal from the E2 catalytic Cys. The C-terminal "C-lobe" contains the HECT catalytic Cys, which receives Ub from the E2 to form a thioester-linked E3~Ub complex. In the only crystal structure of an E2-HECT domain complex, containing UbcH7 and the HECT domain of E6AP, a 41 [unreadable] gap separates the E2 and E3 cysteines. Thus, we are studying structures of HECT E3 complexes in complexes with E2~ubiquitin in order to understand fundamental mechanisms of ubiquitin transfer.